Our previous studies indicated that certain pseudodiploid XX teratocarcinoma stem cell lines contain two genetically active X chromosomes as long as they are maintained in the undifferentiated state and that the cells express the biochemical manifestations of X inactivation when they are allowed to differentiate in vitro. We have now completed a study of the replication patterns of the X chromosomes in these cells and have found that when the cells are maintained in the undifferentiated state both X chromosomes replicate at the same time as the autosomes, whereas when they are allowed to differentiate, one of the two X chromosomes in a large proportion of the cells switches to an allocyclic replication pattern. These data conclusively demonstrate that such teratocarcinoma stem cells can provide a valid model system, as judged by both biochemical and cytogenetic criteria, for the study of X inactivation. We are now using these cells to study the question of whether methylation of specific X DNA sequences is involved in the initiation and/or maintenance of X chromosome inactivation.